Transient granulated lymphocytes are described in the pregnant uteri of >20 species1. In women and mice, these cells are Natural Killer (NK) cells and their activation/maturation depends upon uterine decidualization rather than presence of conceptuses.2,3 The life history and functions of uterine NK (uNK) cells are more fully known in rodents than women. In women, uNK cells are most frequent in first trimester, representing over 70% of the nucleated bone marrow-derived cells in decidual cell suspensions. Human data also suggest that uNK cells are distinctive, tissue based cells. Most circulating human NK cells are CD16+ CD56dim; uNK cells are CD16− CD56bright 2,4. The minor circulating CD56bright subset preferentially expresses (95%) very high levels of L-selectin5, a molecule central to initiation of extravasation. Fewer CD56dim circulating cells (24%) express L-selection and at a much lower surface density5. The two major NK cells functions, target cell lysis and cytokine production, may be displayed separately or dually by single cells6. Human uNK cells display both functions in vitro7-9 but their in vivo functions are undefined. Many current studies of human uNK function address interactions with trophoblast10-12. Other recent reports indicate human uNK cells express angiogenic factors including Ang-213, an antagonist to endothelial cell TIE-2 and thus, a vessel destabilizing molecule and NKG5, a potent endothelial cell mitogen14.
In vivo studies of murine uNK cell functions advanced rapidly after availability of strains genetically deficient in NK cells15, 16. Histological studies established that NK cell deficient mice do not differentiate uNK cells17, 18. In NK-deficient strains with unrelated genetic changes, a common uterine phenotype was developed by 48 hr after implantation. The anomalies were endothelial cell hypertrophy and damage in mesometrially-positioned uterine vessels, lack of uterine arteriole wall and lumen changes indicative of pregnancy and hypocellularity of decidua. Absence of lytic NK cell function does not explain these results, thus, a cytokine deficiency hypothesis was pursued. Interferon-gamma (IFN-γ) is the prototypic cytokine product of NK cells. IFN-γ is an induced molecule known to regulate expression of >1000 genes, many of which are expressed by vascular and decidual tissues19, 20. IFN-γ is expressed in human and murine uteri during normal gestation21-23 but many authors regard IFN-γ as detrimental to pregnancy24, 25. In an experimental series, it was found (i) IFN-γ peaks in mouse mesometrial uterus at gestation day (gd) 10 at 10 IU/implantation site and (ii) only 10% of this comes from non uNK cells26. Transplantation showed that higher levels of uNK-cell derived IFN-γ are essential for pregnancy-induced modification of the spiral arteries and integrity of decidua while the lower level, non lymphocyte-derived IFN-γ is adequate for maturing uNK cells and limiting their numbers. Daily recombinant mulFN-γ (100–1000 IU/6 days) in alymphoid mice promoted full uterine artery modification and normalized deciduas27. Tumor necrosis factor-alpha, another NK and uNK cell product, lacked these effects26, 27.
Mechanisms that transform endometrium to decidua are endocrine-related. In humans, decidualization begins 7–14 days after the surge in luteinizing hormone (LH) (LH+7–14) and continues if pregnancy occurs28-30. Early decidual development appears important for implantation and most human pregnancy wastage occurs in this peri-implantation interval. Human uNK cells begin to increase in number about LH+3, encircling arteries and uterine glands. Stromal cell changes that cuff the spiral arteries (Streeter's columns) are seen at LH+8. By LH+11 to +13, very large numbers of uNK cells are found throughout the stroma accounting of 30–40% of all cells4. Gap junction-like contacts are found between some human uNK cells and early decidua, that appear essential to the continued differentiation of both cell types31, suggesting that normal uNK cell numbers and levels of function contribute to human implantation success.
Reproductive cycles of mice differ to human by virtual absence of a luteal phase. Decidualization and uNK cell activation are initiated by implantation, thus, uNK cell deficits do not influence mouse embryo implantation17, 22. UNK cells proliferate rapidly within decidua2, 3 but recently it was established that self-renewing uNK progenitor cells do not reside there. Uterine segments from normal mice were grafted by end-to-end anastomosis into uNK cell deficient or normal recipients who were then mated. uNK cells were generated only when hosts had NK cell progenitors33,34. In pregnant mammals, thymus and marrow involute during pregnancy35, 36 while spleen and lymph nodes (LN) become hypercellular37, 38. By transplanting to NK cell deficient mice with established pregnancies, it was found that gestation induces acute uNK cell recruitment from spleen but not from marrow or thymus. uNK cells appear to be recruited to decidua basalis and then move into the mesometrial triangle, the entry portal for nerves and vessels supplying the uterus and developing feto-placenta units3,39,40. In decidua basalis of normal mice at mid gestation, 7% of uNK cells are within vessels homologous to spiral arteries, another 30% are in walls of these vessels and the remaining cells, as resolved in paraffin-embedded sections, are associated with other tissues41.
In normal mice, uNK cells are a major source of inducible nitric oxide synthase22, an IFN-γ regulated enzyme producing the powerful vasodilator nitric oxide (NO). In uNK cell deficient mice, expression of this enzyme is induced in trophoblast but at very low levels22 that cannot dilate the spiral arteries (Kiso and Croy, unpublished vascular casting data). Ineffective dilation of spiral arteries is a hallmark of the human gestational complication hypertension/pre-eclampsia42, 43. Despite extensive study of this syndrome, its frequency remains constant and there is no consensus on underlying causes44, 45. Systemic endothelial cell damage underlies clinical symptoms and may be mediated by dysregulated blood cytokine balance46, 47. Some authors suggest immunological contributions48-50 but assessment of changes in frequency or functional activities of uNK cells is just beginning51-53. Women achieving pregnancy by assisted reproductive technology (ART) are reported at higher risks for pre-eclampsia than women carrying naturally conceived conceptuses54-60. Thus, inappropriate uterine recruitment of human NK cells may contribute to two health-related problems: implantation failure through lack of decidual maintenance and predisposition in pregnant women to preeclampsia. Therefore, it is critically important to define the molecules contributing to the movement of human uNK cells and their progenitors into the uterus and to the specification of their intrauterine locations. Subnormal uNK cell frequencies are reported in women with recurrent spontaneous abortion61, suggesting an additional obstetrical group that may benefit from the proposed studies.
Movement of leukocytes from vessels into tissue has been extensively characterized in non-reproductive organs and many techniques have been validated for such work62-65. Lymphocytes constitutively express the tethering molecule L-selectin, which interacts with Peripheral LN Addressin (PNAd) and Mucosal Vascular Addressin-1 (MAdCAM-1) expressed by the microvillous surface of endothelium in LN and Peyers Patches (PP). Avidity of these interactions is modulated by physiological responses including cytokines, inflammation and fever which trigger rolling for egress of non activated lymphocytes from vessels5, 66-68. Firmer adhesion and trans-endothelial migration involve integrins, particularly α4β7, which uses MAdCAM-1. Recruitment of activated cells requires only the latter mechanism and down regulation of L-selectin is paired with upregulation of α4β7 as naive cells begin to roll and dock. In the presence of cytokines, Vascular Cell Adhesion Molecule 1 (VCAM-1) is induced on endothelium and utilized by lymphocytes69. The β2 integrin, Leukocyte Function Associated Antigen-1, (LFA-1) interacting with its ligands Intercellular Adhesion Molecules (ICAM)-1 and -2 also mediates firm adhesion but is not modified by fever ranges similar to those seen at human ovulation68.
In view of the foregoing, there is a need in the art to define the molecules contributing to the movement of human uterine NK cells and their progenitors into the uterus in order to determine if the uterine environment is amenable to sustaining a pregnancy.